Apoptosis, or programmed cell death, is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death can be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (I-V). Wyllie (1980) Nature 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299; Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Kruman et al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991) Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEB J. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267.
While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including but not limited to, cardiovascular disease; cancer regression; immune disorders, including but not limited to systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including but not limited to, diarrhea and dysentery. In the myocardium, apoptotic cell death follows ischemia and reperfusion.
In Alzheimer's disease, Parkinson's disease, Huntington's chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) Human Immunodeficiency Virus (HIV) infection, which induces apoptosis of T-lymphocytes.
In contrast, the level of apoptosis is decreased in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.
In recent years, a family of proteins has been discovered that controls apoptosis. The prototype of this family is Bcl-2, a protein that inhibits most types of apoptotic cell death and is thought to function by regulating an antioxidant pathway at sites of free radical generation. Hockenbery et al. (1993) Cell 75:241-251. More recent data suggests that Bcl-2 can also function as a channel protein and as an adaptor/docking protein. Reed, et al. (1997) Nature 387:773-776. Together, the Bcl-2 family of proteins are important intracellular modulators of apoptosis and can be divided into two groups based on their effect on apoptosis. Thus, in a general sense, Bcl-2, Bcl-xL, Mcl-1, BHRF-1 and E1B19K are cell death inhibitors (anti-apoptotic), while Bak, Bax and Bcl-xS accelerate cell death (pro-apoptotic).
Bcl-2 family members are generally localized to the outer mitochondrial membrane, the nuclear membrane and the endoplasmic reticulum, where they associate with membranes by virtue of their C-terminal hydrophobic tail. All members of the family have two highly conserved regions, called BH1 and BH2, that permit specific interactions between two members to form stable dimers. Their mechanism of action is presently unclear; however, it is known that the ratio of anti-apoptotic to pro-apoptotic Bcl-2 family members in a cell is critical to the cell's survival following initiation of an apoptotic signal.
Proteins that interact with and alter the activity of Bcl-2 have been described. For example, BAG-1 binds Bcl-2, enhances the anti-apoptotic effect of Bcl-2 and furthermore activates Raf-1. Wang et al. (1996) Proc. Natl. Acad. Sci. USA 93:7063-7068; Takayama et al. (1995) Cell 80:279-284. Other proteins with bcl-2 binding activity include the ras-related R-ras p23, BAP and Bad. Fernandez-Sarabia and Bischoff (1993) Nature 366:274-275; U.S. Pat. No. 5,539,085; U.S. Pat. No. 5,539,094; PCT Application WO 96/13614. Identification of such proteins is of great importance, since an understanding of the protein-protein interactions in which apoptosis-related proteins are involved can not only provide insights into the mechanisms of action of these proteins, but can also provide a focal point toward which apoptosis-modulating therapies can be designed. For example, disruption of a protein-protein interaction between an apoptosis-related protein and a protein which enhances its function can decrease the level of apoptosis in a cell. This may be a desired effect in a tissue which displays an inappropriately high level of apoptosis.
Bak is a member of the Bcl-2 family and is expressed in heart and other tissues. Bak protein is capable of either killing cells, or actively protecting cells from cell death, depending on how this protein interacts with other cellular proteins. Bcl-2 family members are extremely important in determining the fate of a cell following an apoptotic signal, and Bak may be the most important in the major organs such as heart. In the treatment of heart disease, viral infection and cancer, modulation of the interactions between proteins that control apoptosis is a major focal point.
Accordingly, there is a need to identify the proteins that operate to control cell death, and to develop therapeutic reagents that modify the actions of those proteins. The present invention relates to a novel Bak binding protein (BBP), the gene encoding the novel protein, methods for detecting substances that alter the specific binding between Bak and BBP, as well as diagnostic and therapeutic methods utilizing BBP. The invention additionally encompasses novel peptides, designated the “BBP Binding Domains” and novel nucleotides, designated “bbpbd-1” and “bbpbd-2” (collectively “bbpbd”) encoding the peptides, which are involved in the interaction between a protein involved in apoptosis and a protein that binds to it.
All references cited herein are hereby incorporated by reference in their entirety.